1. Field of the Invention
The invention encompasses fusion polypeptides capable of binding vascular endothelial cell growth factor (VEGF), VEGF family members, and splice variants with specifically desirable characteristics, as well as therapeutic methods of use.
2. Brief Summary of the Invention
In a first aspect, the invention features an isolated nucleic acid molecule encoding a fusion polypeptide comprising receptor components R1-R2-F, wherein R1 is vascular endothelial cell growth factor (VEGF) receptor component Ig domain 2 of Flt-1 (Flt1D2), R2 is VEGF receptor component Ig domain 3 of Flk-1 (Flk1D3) (also known as KDR), and F is a fusion component.
In a related second aspect, the invention features a VEGF-binding fusion polypeptide comprising VEGF receptor components R1-R2-F, wherein R1, R2, and F are as defined above. The components may be connected directly to each other or connected via one or more spacer sequences. In a preferred embodiment, R1 and R2 are the only receptor components present. In a specific embodiment, the VEGF-binding fusion polypeptide is amino acids 27-129 (R1) and 130-231 (R2) of SEQ ID NO:8, or a variant thereof.
The fusion component F is selected from the group consisting of a multimerizing component, a serum protein, or a molecule capable of binding a serum protein. In a preferred embodiment, F is a multimerizing component capable of interacting with a multimerizing component on another fusion polypeptide to form a multimeric structure, e.g., a dimer or trimer. Most preferably, the F is selected from the group consisting of (i) a multimerizing component comprising a cleavable region (C-region), (ii) a truncated multimerizing component, (iii) an amino acid sequence between 1 to about 200 amino acids in length having at least one cysteine residue, (iv) a leucine zipper, (v) a helix loop motif and (vi) a coil-coil motif. Preferably, the multimerizing component is an immunoglobulin domain. In one embodiment, F is a full-length or truncated immunoglobulin domain consisting of amino acids 232-458, 232-457, or 352-458 of SEQ ID NO:8.
The receptor components may be arranged in different orders, for example, R1R2F; R2R1F; R1FR2; R2FR1; FR1R2; FR2R1, etc. The components of the fusion polypeptide may be connected directly to each other, or connected via a spacer sequence.
In a third aspect, the invention features a multimeric VEGF-binding protein, comprising two or more fusion polypeptides of the invention (also called a VEGF “trap”). A VEGF trap composed of two fusion polypeptides having at least one truncated multimerizing component is termed a “truncated mini-trap.” The multimeric VEGF-binding protein of the invention is capable of binding VEGF with an affinity (Kd) of at least 1×10−10 M, preferably at least 1×10−11 M, even more preferably at least 1×10−12 M, as measured by BIACORE™ based assays.
The C-region may be created in the multimerizing component by insertion, deletion, or mutation, such that an enzymatically or chemically cleavable site is created. The C-region may be created in any multimerizing component and at any position within; preferably, the C-region is created in a full-length Fc domain, or a fragment thereof, or a CH3 domain. The C-region may be a site cleavable by an enzyme, such as, thrombin, ficin, pepsin, matrilysin, or prolidase or cleavable chemically by, for example, formic acid or CuCl2.
In all embodiments of the VEGF-binding fusion polypeptides of the invention (including full length VEGF-binding fusion polypeptides, truncated VEGF-binding fusion polypeptides, etc.), a signal sequence (S) may be included at the beginning (or N-terminus) of the fusion polypeptide of the invention. The signal sequence may be native to the cell, recombinant, or synthetic.
The components of the fusion polypeptide may be connected directly to each other or be connected via spacers. In specific embodiments, one or more receptor and/or fusion partner components of the fusion polypeptide are connected directly to each other without spacers. In other embodiments, one or more receptor and/or fusion partner components are connected with spacers.
In a fourth aspect, the invention encompasses vectors comprising the nucleic acid molecules of the invention, including expression vectors comprising the nucleic acid molecule operatively linked to an expression control sequence. In a fifth aspect, the invention encompasses host-vector systems for the production of a fusion polypeptide which comprise the expression vector, in a suitable host cell; host-vector systems wherein the suitable host cell is a bacterial, yeast, insect, mammalian cell; an E. Coli cell, or a COS or CHO cell. Additional encompassed in a sixth aspect are VEGF-binding fusion polypeptides of the invention modified by acetylation or pegylation, and other post-translational modifications resulting from expression in a mammalian cell line. Methods for acetylating or pegylating a protein are well known in the art. In specific embodiments, the fusion polypeptide of the invention expressed in a mammalian cell line such as a CHO cell comprises amino acids 27-457 of SEQ ID NO:8 and is glycosylated at Asn residues 62, 94, 149, 222 and 308.
In a related seventh aspect, the invention features a method of producing a VEGF-binding fusion polypeptides of the invention, comprising culturing a host cell transfected with a vector comprising a nucleic acid sequence of the invention, under conditions suitable for expression of the protein from the host cell, and recovering the fusion polypeptides so produced.
The VEGF-binding fusion polypeptides of the invention are therapeutically useful for treating any disease or condition which is improved, ameliorated, or inhibited by removal, inhibition, or reduction of VEGF. A non-exhaustive list of specific conditions improved by inhibition or reduction of VEGF include, for example, undesirable plasma leakage or vascular permeability, undesirable blood vessel growth, e.g., such as in a tumor, edema associated with inflammatory disorders such as psoriasis or arthritis, including rheumatoid arthritis; asthma; generalized edema associated with burns; ascites and pleural effusion associated with tumors, inflammation or trauma; chronic airway inflammation; asthma; capillary leak syndrome; sepsis; kidney disease associated with increased leakage of protein; pancreatic ductal adenocarcinoma (PDAC) and eye disorders such as age related macular degeneration and diabetic retinopathy. The VEGF mini-trap is particularly useful in treatment of eye disorders, and as an adjuvant to eye surgeries, including glaucoma surgery; and the treatment of intra-ocular tumors, such as for example, uveal melanoma, retinoblastoma, via intravitreal delivery.
Accordingly, in an eighth aspect, the invention features a therapeutic method for the treatment of a VEGF-related disease or condition, comprising administering a VEGF-binding fusion polypeptide of the invention to a subject suffering from a VEGF-related disease or condition. Although any mammal can be treated by the therapeutic methods of the invention, the subject is preferably a human patient suffering from or at risk of suffering from a condition or disease which can be improved, ameliorated, inhibited or treated with a VEGF-binding fusion polypeptide of the invention.
In a ninth aspect, the invention features pharmaceutical compositions comprising a VEGF-binding fusion polypeptide of the invention with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may comprise a dimeric fusion polypeptide trap, or nucleic acids encoding the fusion polypeptide. The mini-traps of the invention find specific uses in conditions in which a VEGF inhibitor with reduced serum half life (e.g., faster clearance), and/or increased tissue penetration due to smaller size is desirable. Specific applications for the VEGF mini-trap include, for example, diseases where local administration to a specific tissue or cell is desirable. Examples of such a condition or disease are ocular diseases of the eye.
Other objects and advantages will become apparent from a review of the ensuing detailed description.